Image ver 1

The activity of matrix metalloproteinases (MMPs) was detected by gelatin zymography using Novex Zymogram Gels, Tris-Glycine SDS Sample Buffer, Zymogram Renaturing Buffer, and Zymogram Developing Buffer (Invitrogen, Carlsbad, CA). In brief, after lung tissue proteins (50 µg) of soluble and precipitated fractions were electrophoretically separated on gels, the gels were incubated in renaturing buffer at room temperature for 1 h and then in developing buffer at 37°C for 16 h. To stop the reaction, a specific protease inhibitor was added to the developing buffer. Quantitative analysis of the gelatinolytic enzyme was performed using Scion Image ver. 1.63.

Frozen liver tissues were homogenized in sample buffer (50 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10 mM dithiothreitol, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged at 12,000 g at 4°C for 15 min. Proteins (30 µg) of soluble and precipitated fractions were electrophoretically separated by 7.5–15% SDS-polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then incubated with an anti-desmin polyclonal antibody (Abcam, Cambridge, UK), anti-α-SMAβ1 monoclonal antibody (Oxford Biomedical Research, Oxford, MI), anti-tissue Inhibitor of metalloproteinase 1 (TIMP-1) monoclonal antibody (R&D Systems, Minneapolis, MN), or anti-collagen-1 antibody (Abcam). Membranes were then incubated with secondary anti-rabbit, -mouse, or -goat IgG (Dako Cytomation; Kyoto, Japan), followed by incubation for 2 min with ECL solution and then exposure to X-ray film. Western blots were quantified using Scion Image ver. 1.63.